Characterization of antimicrobial resistance pattern and genes in Klebsiella species isolated from domestic poultry and farm environment

Introduction

The global antibiotic consumption in animals is estimated to be twice that of humans, and the improper and excessive use of these drugs in veterinary practices, both as therapeutic and growth promoter agents, has contributed significantly to the emergence of multidrugresistant (MDR) microorganisms. This issue is particularly concerning in modernized intensive poultry farming systems, where large populations of birds are kept in close vicinity, creating an environment that fosters the spread of resistant bacteria (Chaudhry et al., 2020) [1,2] . Among the most problematic pathogens associated with antibiotic resistance is the ESKAPE group, which includes Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, and Enterobacter species. These pathogens are notorious for causing nosocomial infections and are known for their ability to "escape" the effects of antimicrobial treatments. Klebsiella pneumoniae is particularly concerning due to its role in severe infections, especially in immunocompromised and critically ill patients, and its increasing resistance to antibiotics (Singh, 2018) [3] . It also poses significant risks in animal populations, particularly poultry. The upsurge of antibioticresistant Klebsiella pneumoniae strains presents a major challenge in both veterinary and human medicine. Over the past few years, K. pneumoniae has developed resistance to a broad range of antibiotics, including β-lactams, aminoglycosides, carbapenems and fluoroquinolones. The primary mechanism behind this resistance is the production of extended-spectrum β-lactamases (ESBLs) and carbapenemases enzymes that break down antibiotics and render them ineffective. Carbapenems, which are often used as a last-resort treatment for severe infections, are becoming increasingly ineffective against K. pneumoniae, raising serious concerns about the future of treatment options (El-Sukhon, 2003) [4] . The overuse of antibiotics in both human medicine and animal agriculture has accelerated this trend, making it a pressing public health issue. Given the growing threat of MDR K. pneumoniae, understanding the sources and mechanisms of resistance in these bacteria is crucial. This study aimed to achieve two primary objectives: 1) to analyze the antimicrobial resistance patterns exhibited by the Klebsiella isolates, and 2) to detect specific antimicrobial resistance genes (ARGs) in these isolates. These objectives were designed to provide insight into the genetic factors contributing to resistance and to assess the potential risk of transmission from animals to humans. This research is critical for understanding the spread of MDR Klebsiella in domestic poultry and its surrounding environment. By identifying resistance patterns and detecting resistance genes, this study provides valuable information that can inform future strategies to control the spread of MDR bacteria in both veterinary and human healthcare settings. Understanding how Klebsiella species acquire resistance will aid in developing measures to mitigate the risk of antibiotic resistance spreading from animals to humans, ultimately contributing to better public health outcomes.

Materials and Methods

The study aimed to isolate Klebsiella species from poultry and the farm environment, assess antibiotic resistance patterns, and detect antimicrobial resistance genes (ARGs). A total of 150 samples were collected from various poultry farms around Mhow and Indore. Out of these 150 samples 17 came positive with phenotypic and genotypic methods. Antimicrobial sensitivity testing was performed using the disk diffusion method on Muller-Hinton agar (Bauer et al., 1966) [5] . Antibiotics used in the present study were: ampicillin/ sulbactum, cefotaxime/ clavulanic acid, cefazolin, ciprofloxacin, tetracycline, ertapenem, amoxyclav, aztreonam, ticarcillin, fosfomycin, chloramphenicol, gentamicin, ofloxacin, amikacin and tigecycline. The presence of ARGs was determined through PCR using specific primers for bla TEM, bla SHV, bla DHAM, sul1, aadA and tetA genes (Table 1), followed by gel electrophoresis to analyze the amplified DNA products. Thermocyclic conditions of each gene is given in table 2.

Results

Antimicrobial resistance pattern in Klebsiella isolates In vitro antibiotic sensitivity

In our study, all Klebsiella isolates were tested against 15 antibiotics using the disc diffusion method (Figure 1). Sensitivity results were interpreted according to the manufacturer's guidelines (Hi Media) and are detailed in Table 3 and Figure 2. The highest sensitivity was observed for amikacin (82.35%), followed by ampicillin-sulbactam (58.82%), ticarcillinclavulanic acid, and ofloxacin (41.17% each). Notably, none of the isolates were sensitive to tigecycline, ciprofloxacin, cefazolin, or cefotaximeclavulanic acid. The highest intermediate sensitivity was recorded for amoxyclav (52.94%) and gentamicin and ertapenem (47.05% each). In the study, resistance to ampicillin-sulbactam and amoxyclav was found to be 23.52% among Klebsiella isolates, while a higher resistance rate of 41.17% was observed for the carbapenem antibiotic ertapenem. Similarly, 23.52% of the isolates were resistant to the monobactam aztreonam. Notably, cefotaxime-clavulanic acid, a cephalosporin, showed no sensitivity towards the tested isolates.

Ciprofloxacin, a third-generation fluoroquinolone, had a high resistance rate of 82.35%, particularly in ESBL-positive strains, a trend consistent with our findings. Additionally, 70.58% of Klebsiella strains were resistant to tetracyclines, while resistance to aminoglycosides, such as amikacin and gentamicin, was 11.76% and 35.29%, respectively. Resistance to chloramphenicol was observed in 35.29% of the isolates, and importantly, all isolates were 100% resistant to tigecycline. In the present study six isolates of Klebsiella spp were found to be multiple drug resistant (MDR), as they showed resistance to at least one antimicrobial agent (Table 4) in three antimicrobial categories as per Sweeney et al. (2018) [6] . Out of 6 MDR strains, 3 were K. pneumoniae, which suggests this bacteria is an emerging multidrugresistant (MDR) pathogen.

Antibiotics Remarks AK AMC A/S AT CEC CZ C CIP ETP FO GEN OF TE TCC TGC

Detection and prevalence of antimicrobial resistance genes in Klebsiella isolates

PCR analysis of DNA from 17 Klebsiella isolates identified the bla TEM gene in 16 isolates (94.11%) through a 506 bp amplicon (Figure 3), while the bla SHV gene was detected in 15 isolates (88.23%) with a 628 bp product (Figure 4). The bla DHAM gene, marked by a 405 bp amplicon, was present in only 2 isolates (11.78%) (Figure 5). The sul1 gene was found in 3 isolates (17.6%) with an 822 bp amplicon (Figure 6). Additionally, the aadA gene (Figure 7) associated with aminoglycoside resistance, was present in 7 isolates (41.17%) with a 284 bp product, and the tetA gene, linked to tetracycline resistance, and was also detected in 7 isolates (41.17%) with an 887 bp product (Figure 8). The prevalence of antimicrobial resistance genes (ARGs) is depicted in the form of a pie chart in Figure 9 and table 5 summarizes the diferent sample sources in which these genes were detected.

Discussion

Increasing antimicrobial resistance is a significant public health issue, with scientists predicting it could lead to the next pandemic. The treatment of infections caused by Gramnegative bacteria, such as Klebsiella, poses a major challenge, especially given the indiscriminate use of antibiotics in livestock and poultry industries, which contributes to the emergence of drug-resistant bacteria (Exner et al., 2017) [7] . In our study, all Klebsiella isolates were tested for susceptibility to 15 antibiotics using the disc diffusion method. The highest sensitivity was observed for amikacin (82.35%), while no isolates were sensitive to tigecycline, ciprofloxacin, cefazolin, or cefotaxime/clavulanic acid. Our findings align with previous studies by Younis et al. (2016) [8] , Akinbami et al. (2018) [9] , and others, which also reported Klebsiella strains sensitive to amikacin but resistant to ampicillin, amoxyclav, aztreonam, and cefotaxime. However, our study contrasts with Diagbouga et al. (2016) [10] , who reported high sensitivity to fosfomycin, a finding not supported by our results. The importance of conducting antibiotic sensitivity testing (AST) prior to treating Klebsiella infections is underscored by the potential for changes in drug sensitivity over time, as highlighted by Aly et al. (2014) [11] Antimicrobial resistance pattern in Klebsiella isolates Antimicrobial resistance has emerged in zoonotic enteropathogens, commensal bacteria, and animal pathogens, with varying prevalence (Carattoli, 2008) [12] . Factors influencing antibiotic resistance include bacterial resistosomes, misuse of antibiotics as therapeutic or prophylactic agents, improper dosing, and the use of antibiotics as growth promoters in livestock (Watkins and Bonomo, 2016; Van Boeckel et al., 2015) [13,14] . In our study, the high degree of resistance observed may be due to the misuse of antibiotics in poultry feed, as also noted by El Tawab et al. (2018) [15] . The prolonged use of low-dose antibiotics can lead to the survival of resistant strains, which eventually dominate the population, contributing to the widespread distribution of antimicrobial resistance (Woodford et al., 2011) [16] Resistance to beta lactam antibiotics Extended spectrum beta-lactam (ESBL) antibiotics, including penicillins, cephalosporins, cephamycins, carbapenems, and monobactams, are widely used to treat serious Gram-negative infections. However, bacterial resistance has emerged due to the production of ESBL enzymes, which deactivate these antibiotics by hydrolyzing the betalactam ring (Bajpai et al., 2017) [17] . In our study, resistance to ampicillin/sulbactam and amoxyclav was observed at 23.52%, which is lower than rates reported by Younis et al. (2016) [8] and others. Klebsiella species naturally resist penicillins due to low production of a class A chromosomal β-lactamase, but resistance can increase when ESBL genes are acquired (Brisse and Duijkeren, 2005) [18] . Ertapenem resistance was high at 41.17%, aligning with Hamza et al. (2016) [19] , highlighting a growing concern of carbapenem resistance even though these drugs are not typically used in poultry. Aztreonam resistance was 23.52%, similar to findings by Tewari et al. (2019) [20] . No sensitivity to cefotaxime/clavulanic acid was observed, consistent with previous studies (Younis et al., 2016; Elgaos et al., 2019) [8,21] . Increasing resistance to cefotaxime, a critical thirdgeneration cephalosporin, may be due to its frequent use in veterinary medicine, as noted in recent reports.

Resistance to quinilones and fluoriquinilones

Fluoroquinolones are critical antibiotics for managing bacterial infections (Cattoir and Nordmann, 2009) [22] . In our study, resistance to ciprofloxacin, a third-generation fluoroquinolone, was observed at 82.35%, which aligns with findings by Diagbouga et al. (2016) [10] , Younis et al. (2016) [8] , and others, although Elgaos et al. (2019) [21] reported a much lower resistance rate of 20%. In India, resistance to fluoroquinolones among Klebsiella spp. has risen from 57% to 73% (CDDEP, 2015). This resistance is often due to chromosomal mutations in quinolone resistance-determining regions (QRDR) and plasmid-mediated mechanisms (Correia et al., 2017) [23] , with higher resistance observed in ESBL-positive strains, as seen in our study. The increasing use of fluoroquinolones in poultry farming, including as growth promoters, may be contributing to the development of resistance, a trend confirmed by veterinarians in the study area (Mahanti et al., 2018) [24] .  Resistance to Tetracyclines: In our study, 70.58% of Klebsiella strains were resistant to tetracyclines, consistent with findings by Bokaeian et al. (2014) [25] and Kashefieh et al. (2021) [26] . Tetracycline resistance is often mediated by ribosomal protection, efflux pumps, or chemical modifications (Moradigaravand et al., 2017) [27] . The widespread use of tetracycline in poultry farms, including in India, due to its broadspectrum efficacy and low cost, may contribute to this resistance (Michalova and Schlegelova, 2004) [28] .  Resistance to Aminoglycosides: Resistance to aminoglycosides like amikacin and gentamicin was observed at 11.76% and 35.29%, respectively, aligning with the findings of Mokhtari et al. (2018) [29] and Kashefieh et al. (2021) [26] . Aminoglycoside resistance mechanisms include enzymatic modification and changes in membrane permeability (El-Badawy et al., 2017) [30] . These drugs are less commonly used in farming due to their cost, which may explain the lower resistance observed (Sidibe et al., 2019).  Resistance to Phenicols:Chloramphenicol resistance in Klebsiella was 35.29% in our study, similar to Younis et al. (2016) [8] . Chloramphenicol resistance is often due to acetylation by chloramphenicol acetyltransferases (Schwarz et al., 2004) [31] , and its use has declined due to associated side effects.  Resistance to Phosphonic Acid:Fosfomycin resistance was very high at 94.11% in our Klebsiella isolates, a concerning finding given its limited use in veterinary practice. This high resistance may be due to horizontal gene transfer from other bacteria (Akinbami et al., 2018) [9] .  Resistance to Glycylcyclines: All Klebsiella isolates in our study were 100% resistant to tigecycline, which contrasts with the 9% resistance reported by Kashefieh et al. (2021) [26] . Tigecycline resistance in Klebsiella is often due to efflux pumps, particularly RND-type transporters (Sun et al., 2013) [32] .

Simultaneous and multiple drug resistance

All 17 isolates showed resistance to 5 to 14 antibiotics, with 64.70% (11 isolates) resistant to 8 to 13 antibiotics, although none were resistant to all 15. These results align with Brower et al. (2017) [33] . Six isolates of Klebsiella spp. were identified as multidrug-resistant (MDR), showing resistance to at least one agent in three different antimicrobial categories, according to Sweeney et al. (2018) [6] . Notably, three of these MDR strains were K. pneumoniae, highlighting its role as an emerging MDR pathogen, consistent with findings by Navon Venezia et al. (2017) [34] and others. This emphasizes K. pneumoniae as a significant contributor to antibiotic resistance, classified as an ESKAPE organism (Boucher et al., 2009) [35] .

Detection of antimicrobial resistance genes in Klebsiella isolates

Under antibiotic pressure, Klebsiella rapidly accumulates antibiotic resistance genes (ARGs) through mutations and acquisition of plasmids and mobile genetic elements. This resistome, the collection of all resistance genes, is often carried on self-transferable plasmids within K. pneumoniae, contributing to the development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains (Navon-Venezia et al., 2017) [34] . In our study, we detected the bla TEM and bla SHV genes in 94.11% and 88.23% of Klebsiella isolates, respectively, with bla TEM being the most prevalent, aligning with findings from previous studies (Fortini et al., 2011; Bajpai et al., 2017) [36,17] . Additionally, the aadA and tetA genes, responsible for aminoglycoside and tetracycline resistance, were present in 41.17% of isolates, consistent with other reports (Kashefieh et al., 2021) [26] . The emergence of these resistance genes highlights the significant role of K. pneumoniae in spreading antibiotic resistance, especially in settings with high antibiotic use. The rise of antibiotic resistance in livestock production is a critical issue, with improper antibiotic use closely linked to the development of AMR in humans (Ozawa et al., 2012) [37] . This study showed a moderate correlation between phenotypic and genotypic resistance in Klebsiella strains, with phenotypic resistance to amoxyclav and ampicillin (82.30%) aligning with the presence of bla TEM and bla SHV genes (94.11% and 88.23%, respectively). Resistance to aminoglycosides (47%) also correlated with the aadA gene (41.17%). Effective surveillance and characterization of antimicrobial-resistant bacteria in livestock and poultry are essential (Tewari et al., 2019) [20] .

Conclusion

This study highlights the significant presence of antimicrobial-resistant Klebsiella species in poultry farms, with a 11.3% prevalence across samples and the highest concentration in farm soil (50.5%). Notably, Klebsiella pneumoniae was identified as the most pathogenic, found in 8% of samples. High resistance rates to ciprofloxacin (82.35%) and tigecycline (100%) were observed, with bla_TEM and bla_SHV genes detected in 94.11% and 88.23% of isolates, respectively. The findings emphasize the need for stringent antibiotic management and continuous monitoring to prevent the spread of multidrug-resistant bacteria, protecting both veterinary and human health.

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